Abstract Sickle cell disease (SCD) is a genetic blood disease in which hemoglobin pathologically polymerizes in red blood cells (RBC). This results in elongation of RBCs (the ?sickle? shape) and increased RBC stiffness that subsequently causes poor blood flow followed by life-threatening organ damage, which typically occurs in the low oxygenated venous circulation. However, newer research has definitively demonstrated that SCD is much more complex than originally thought. For example, almost of the blood vessels in SCD patients, in the arterial as well as venous circulations, are known to be dysfunctional and prone to inflammation, which in turn, predisposes patients to acute and poorly understood complications such as stroke. Recent work has also demonstrated that all blood cells including RBCs, white blood cells, and platelets pathologically adhere and interact with endothelial cells, the cells that line the inner wall of blood vessels, which contribute to the pro- inflammatory state of the endothelium. However, this likely does not account for all of the endothelial dysfunction seen in SCD and as such, this pathological process remains poorly understood. For this R21 grant, the bioengineering hematology laboratory of Wilbur Lam, MD, PhD and the computational fluid dynamics lab of Michael Graham, PhD, will continue their longstanding collaboration to investigate a novel hypothesis: that the stiffened RBCs in SCD are constantly colliding with endothelial cells to induce cell dysfunction. We base this hypothesis on two well-documented biophysical phenomena described by labs including our own: 1) blood cells predominantly flow in the center of blood vessels and but are driven towards the endothelial cells (i.e., cell margination) when they become stiffer (as is the case in SCD), and 2) endothelial cells ?feel? physical forces and biologically respond to aberrant forces by activating inflammatory pathways. As these biophysical variables are too difficult to control in in vivo SCD animal models, to explore our hypothesis, we will analyze SCD patient blood samples in in vitro ?endothelialized? microvasculature-on-a-chip devices developed by the Lam Lab coupled with the computational modeling techniques developed by the Graham Lab. Specifically, we will determine the effects of perfusing SCD RBCs of different shapes and stiffnesses on endothelial function, as measured with antibody staining of cellular markers of inflammation, using our in vitro vasculature models coupled with complex single cell computational modeling to gain systematic and mechanistic insight into our experimental data. Furthermore, we will also assess how the hemodynamic flow pattern (different flow rates with steady versus pulsatile flow) and complex vessel geometry (e.g. size, curvature, bifurcation) affect SCD RBC margination and therefore endothelial pro-inflammatory pathways. Overall, this work will determine whether purely non-adhesive, physical interactions between endothelial cells and SCD RBCs are sufficient to cause endothelial dysfunction, which may help explain why SCD patients develop stroke and may lead to a new paradigm of physics-based diagnostic and therapeutic strategies for SCD.